Large amounts of glycerol are produced as a by product in the production of biodiesel, and the production of glycerol is predicted to continue to increase because of the tremendous global growth in the production of biodiesel. The current surplus of glycerol has already resulted in a ˜10 fold reduction in price in the last two years.
Crude glycerol, therefore, has become an attractive carbon source for fermentation processes, in part because of its low cost. Not only is glycerol abundant, but its higher reduced state, compared to cellulosic sugars, promises to significantly increase the product yield of chemicals whose production from sugars is limited by the availability of reducing equivalents. Taking advantage of the higher reduced state of carbon in glycerol would require the use of anaerobic fermentations (i.e., otherwise the electron acceptor will “take” the electrons instead of being “deposited” in the desired product, including fuels or reduced compounds). The potential use of glycerol as carbon source in fermentation processes could be hampered, however, by the inability of industrial microorganisms, such as Escherichia coli (the workhorse of modern biotechnology) to ferment glycerol in the absence of external electron acceptors. The ability to ferment glycerol is restricted to very few organisms, most of them not amenable to industrial applications due to pathogenicity, requirement of strict anaerobic conditions, lack of physiological knowledge and genetic tools for their modification, and high nutritional requirements.
Glycerol can be directly oxidized by the enzyme glycerol dehydrogenase (GldA, encoded by gldA) generating dihydroxyacetone (DHA). However, GldA, which is characterized as a type II glycerol dehydrogenase (glyDH-II), is thought to be cryptic or not expressed in wild type E. coli strains. Activation of GldA in E. coli required inactivation of glpK, glpR, and glpD followed by a mutagenesis and selection procedure which resulted in a mutant strain that recovered its ability to metabolize glycerol (Jin et al., 1983; Tanh et al., 1982a, b). However, even in this mutant strain, GldA did not provide E. coli with the ability to fermentatively metabolize glycerol (Jin et al., 1983; Tanh et al., 1982a, b).
What is needed in the art is a method of bioconverting the global surplus of inexpensive glycerol into other feedstock chemicals. The method would be specially advantageous should it be based on anaerobic fermentation to take advantage of higher reduced state of carbon in glycerol. An anaerobic process would also offer cost advantages, as their counterpart aerobic processes require higher capital investment and exhibit higher operational costs.